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Abstract

Background

Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared
the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate
the prevalence of Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and
to identify potential determinants of the disease and hotspots of malaria transmission
within the country.

Methods

The prevalence of P. falciparum and P. vivax within the districts of the capital city and the rest of the Republic of Djibouti
were assessed using 13 and 2 serological markers, respectively. The relationship between
the immune humeral response to P. falciparum and P. vivax and variables such as age, gender, wealth status, urbanism, educational level, distance
to rivers/lakes, living area, having fever in the last month, and staying in a malaria-endemic
country more than one year was estimated and analysed by questionnaires administered
to 1910 Djiboutians. Multivariate ordinal logistic regression models of the immune
humeral response were obtained for P. falciparum and P. vivax.

Results

The P. falciparum and P. vivax seroprevalence rates were 31.5%, CI95% [29.4-33.7] and 17.5%, CI95% [15.8-19.3],
respectively. Protective effects against P. falciparum and P. vivax were female gender, educational level, and never having visited a malaria-endemic
area for more than one year. For P. falciparum only, a protective effect was observed for not having a fever in the last month,
living more than 1.5 km away from lakes and rivers, and younger ages.

Conclusions

This is the first study that assessed the seroprevalence of P. vivax in the Republic of Djibouti. It is necessary to improve knowledge of this pathogen
in order to create an effective elimination programme. As supported by recent observations
on the subject, the Republic of Djibouti has probably demonstrated a real decrease
in the transmission of P. falciparum in the past seven years, which should encourage authorities to improve efforts toward
elimination.

Keywords:

Background

According to the World Health Organization (WHO), approximately 3.3 billion people,
nearly half of the world population, are at risk of malaria. Each year, approximately
250 million people contract the disease, and nearly one million people die. The inhabitants
of the poorest countries are the most vulnerable. More than one in five infant deaths
(20%) occurring in Africa are due to malaria
[1]. However, the number of Plasmodium falciparum malaria cases is declining, even in Africa. Given this situation, in the late 1990s,
WHO proposed a goal of controlling the disease and achieving elimination by 2015 in
areas of low transmission. Policies, international and national initiatives have proliferated
to help the neediest.

Based on the results of scientific research in all areas of malaria control and because
of greater knowledge of the disease and its medical and social consequences, the proposed
strategy is organized into two main phases: control and disposal
[2]. Among the actions undertaken on a large scale, it should be noted that the availability
and distribution of ITNs and ACT, vector control through IRS, active detection of
new breeding sites and their systematic destruction represent a link essential to
the success of disease control before disposal is considered
[3].

According to the Roll Back Malaria project, malaria primarily concerns 109 countries,
but 35 countries account for 98% of malaria deaths worldwide. Only five of these countries
(Nigeria, Democratic Republic of Congo, Uganda, Ethiopia and Tanzania) represent 50%
of deaths and 47% of malaria cases
[4]. Among these countries, Ethiopia and Uganda share an economic community, bringing
together 340 million people who are free to move to the Republic of Djibouti
[5].

Formerly known to be a malaria meso- to hypoendemic country with an unstable malaria
transmission profile
[6-8], this country of 818,159 inhabitants declared a goal of malaria pre-elimination in
2006
[9]. Micro-epidemics can occur in the presence of favourable set of conditions, such
as unusual rainfall (the last major outbreak occurred in 1999)
[10,11]. Over the last 14 years, the transmission and the malaria cases number remained low.
As a result, foreign armies present in the Republic of Djibouti have recently considered
stopping their malaria chemoprophylaxis
[12], as the French army did last year.

Djibouti has recently demonstrated its eligibility for the pre-elimination goal according
to technical feasibility, i.e., the baseline domestic malaria transmission combined
with the importation-related transmission and operational feasibility, which takes
into consideration the country government status, health status and information on
populations at risk
[13]. These observations are in agreement with parasite genetic diversity studies
[10,11], and one recent work has reported a low transmission level
[14].

According to WHO, malaria control requires, at a national level, the expertise and
development of databases containing information about the parasites found locally
and information about changes in transmission levels and the status of resistance
to anti-malarials
[15]. Control in the short- and medium-term is possible by developing constantly improved
detection and observation tools and early care and adequate diagnoses in risk populations
[15].

Therefore, serological tools are widely used to assess the transmission level and
thus the prevalence of Plasmodium falciparum and Plasmodium vivax in human populations and to assess epidemiological facts of the past and present
[16,17]. One recent work in Somaliland (the nearest neighbouring country to Djibouti with
regular movements of the population in both directions) has used serological tools
to assess the prevalence of P. falciparum and P. vivax[18-20].

Starting from this observation, it was necessary to perform a similar survey in the
Republic of Djibouti. Because most of the previous studies have primarily concerned
P. falciparum malaria, it was also necessary to study P. vivax malaria and mixed infections to gather enough information for elimination
[3].

The aim of the present study was to evaluate the prevalence rate of P. falciparum, P. vivax and mixed infections in the Djiboutian population by using serological tools and
to identify potential determinants of hot spots of malaria infection and transmission
within the country.

Methods

Sera samples

The prevalence rate of P. falciparum and P. vivax infections among adults aged 15–54 years living in the Republic of Djibouti was estimated
using a anonymous non-correlated cluster sampling method between the 24th and the 31th of March 2002. In brief, 30 clusters were investigated in the city of Djibouti, and
25 clusters in the other districts of the country. The clusters were randomly selected
proportionally to the population size according to the list of quarters used by the
National Direction of Statistics in the city of Djibouti and the list of the towns
used by the expanded programme of immunization in the other districts. In each selected
site, a starting household was randomly selected, and the next nearest households
were investigated until a total of 44 resident adults per cluster in the city of Djibouti
or 35 resident adults per cluster in the other districts were obtained. A total of
1,910 blood samples were collected anonymously in accordance with the recommendations
of the Djiboutian Ministry of Health, who gave the ethics clearance for the present
study. Blood samples were stored at 4 °C until separation of plasma by centrifugation
(less than 24 hours after collection) and freezing. Thirty sera samples from French
adults who had never been to malaria-endemic countries were used as unexposed negative
controls. For the seropositivity threshold, the means and standard deviations (SDs)
of the antibody intensity of the negative control group for all antigens were estimated.
The lower limit of positivity for each antibody was taken as the mean + 3 SD of the
negative control group values and corresponded to a mean fluorescence intensity (MFI)
of 1,000. For P. falciparum, a sample was considered to be positive if the reactivity to at least two different
plasmodial antigens was > 1,000 MFI. For P. vivax, a sample was considered to be positive if the reactivity to PvMSP1-19 or to PvMSP1-42
was > 1,000 MFI.

Bead-based assay

Peptides and proteins were coupled to beads as described by Ambrosino et al.
[22], and an optimal concentration of 0.3 nmol was used for each antigen. Furthermore,
BSA coated beads were included as a background control. Ag-coated beads were resuspended
by vortexing and sonication for 5 minutes and were diluted in equal volumes of PBS
and MFIA (Multiplexed Fluorescence ImmunoAssay) diluent (Charles River Laboratories
Inc, MA, USA) to a final concentration of 80 beads/μl per peptide. The 1.2-μm filter-bottom
96-well microtiterplates (MSBVS 1210, Millipore, MA, USA) were rewetted with washing
buffer (0.15% Tween 20 in PBS 7.4) using a vacuum manifold (Millipore). Fifty microliters
of beads and sera (diluted 1:100 in equal volumes of PBS and MFIA diluents) were added
to each well. Plates were incubated at room temperature in the dark for 1 h with shaking
at 600 rpm. After incubation, plates were washed eight times with 200 μl of washing
buffer, then 100 μl of the secondary Ab (R-phycoerythrin F(ab’)2 fragment of goat
anti-human IgG, (Interchim, Montluçon, France), diluted 1:500, was added to each well.
After 30 min of incubation in the dark at room temperature with shaking, plates were
washed as described previously. Beads were resuspended in 100 μl of a solution of
5% BSA-PBS, pH 7.4 and analysed on Luminex system. The system was set to read a minimum
of 100 beads per spectral address, and the results were expressed as MFI.

Data collection

During the cross-sectional study between the 24th and the 31th March 2002, self-administered questionnaires containing several items were filled
out by Djiboutian inhabitants and validated by a member of the research team. Different
types of independent variables were collected: the living area (the city of Djibouti
or the rest of the country), the type of living area (urban or rural), having stayed
in a malaria-endemic country for more than one year (yes or no), having had a fever
during the last month before the study (yes or no), the utilization of bed nets (often
to always and rarely to never), gender (male or female), schooling status (schooled
or never schooled), educational level (never schooled, primary school, secondary school,
high school or university), wealth (poor = less than 65,000 Djiboutian Francs per
home and rich = more than 65,000 Djiboutian Francs per home), and age.

A Geographic Information System was built in ArcGIS 9.2 (Environmental Research Systems
Institute, Redlands, CA). The layers were added as follows: i) map of inland water
in Djibouti and the neighbouring countries (data from Digital Chart of the World,
accessed through DIVA-GIS
[23] and ii) 60 points corresponding to the sampling locations. At every point, the Euclidian
distance to the first pixel of water was computed. This enabled the creation of a
geographical independent variable, the distance of a cluster to a river or a lake
(≤ 1.5 Km or > 1.5 Km).

Statistical methods

Data were recorded using Excel and were checked for consistency before statistical
analysis using R software (version 2.10.1) or STATA software (version 11). The seropositivity
to P. falciparum antigens, P. vivax antigens or both of them (mixed infection) were analysed as a dependant variable
according to individual and cluster characteristics using a random effect mixed logistic
regression model. The model was designed to take into account the intracluster correlations
that could exist due to the sampling design (cluster effect as random effect). The
logistic model was also adjusted using a generalized estimating equations (GEE) approach.
Random effect and GEE regression models allow the estimation of cluster-specific and
population-averaged effects, respectively
[24]. First, a descriptive analysis of the independent variables was performed. A bivariate
analysis was then conducted by entering each independent variable in a logistic regression
model, and all the results were presented in Additional files
1,
2,
3,
4,
5 and
6. Variables were retained for the multivariate analysis if their effect had a p-value
less than 0.25
[25]. A backward stepwise selection procedure was applied to retain the significant (p < 0.05)
independent variables and their interactions in the final model. The statistical quality
of the final model was assessed by looking at the adequacy between observed and predicted
prevalence rates.

Results

Plasmodium falciparum seroprevalence

The serological analysis showed that 25.90% of sera were positive for at least two
of the following 11 P. falciparum peptides: Lsa1-41, Lsa1-J, Lsa3-NR2, Glurp, GlurpP3, Salsa1, Salsa2, Trap1, Starp-R,
CS (NANP) and SR11.1. The proportion of sera that were positive for both and at least
one of the two recombinant falciparum antigens, i.e., PfMSP1 and PfAMA1, were 13.24% and 29.98%, respectively. By taking
into account the immune humeral response to at least two different peptides or recombinant
proteins of the 13 P. falciparum antigens used in this study, the seropositivity rate to P. falciparum infection was 31.5% (602/1910 Djiboutian people), CI95% (29.4-33.7).

Considering the potential determinants of the P. falciparum malaria infection, according to the results of the multivariate logistic regression
analysis (Table
1), some factors were significantly and independently statistically associated with
a lower risk of being seropositive for P. falciparum:

In contrast, only one risk factor for seropositivity for P. falciparum was identified: older age (40 years to 55 years). There was a non-significant association
of living near lakes and rivers (≤ 1.5 Km) with a higher risk of seropositivity.

Plasmodium vivax seroprevalence

By taking into account the reactivity against PvMSP142 or PvMSP119, the global seropositivity rate to P. vivax was 17.5% (334/1910 Djiboutian people), CI95% (15.8-19.3). Considering the potential
determinants of the P. vivax malaria infection, according to the results of the multivariate logistic regression
analysis (Table
2), some factors were significantly and independently statistically associated with
a lower risk of being seropositive for P. vivax:

– The gender (female gender).

– Never having visited a malaria-endemic country for more than one year.

Having no schooling was significantly and independently statistically associated with
a higher risk of being seropositive for P. vivax infection.

Mixed infection seroprevalence

The global seropositivity rate of mixed infection was 10.2% (195/1910 Djiboutian people),
CI95% (8.9-11.7).The results of the multivariate logistic regression analysis for
predicting a mixed infection (Table
3), i.e., seropositivity to serological markers of both P. falciparum and P. vivax, showed that some variables were significantly and independently statistically associated
with a lower risk of being seropositive for mixed infection:

– The gender (female gender).

– Never having visited a malaria-endemic country for more than one year.

Figure 2.Distribution of corrected MFI values against the P. falciparum antigens in the L1, L2 and L3 groups.

According to the results of the Bayesian ordinal multinomial logistic regression analysis
(Table
4), some factors were significantly and independently statistically associated with
a lower risk of having a high level of immune humeral response to P. falciparum antigens:

– The gender (female gender).

– The educational level (primary school, secondary, high school or university).

– Never having visited a malaria-endemic country for more than one year.

The proportions of P. vivax seropositives to the different recombinant proteins from the different L-groups (L0 = seronegativity
MFI < 1,000, L1 = 1,000 ≤ MFI < 2,000, L2 = 2,000 ≤ MFI < 10,000 and L3 = MFI ≥ 10,000)
were illustrated in Figure
1. The distribution of corrected MFI values against the P. vivax antigens in the L1, L2 and L3 groups was presented in Figure
3. According to the results of the Bayesian ordinal multinomial logistic regression
analysis (Table
5), some factors were significantly and independently statistically associated with
a lower risk of having a high level of immune humeral response to P. vivax antigens:

– The gender (female gender).

– Never having visited a malaria-endemic country for more than one year.

– Not having had a fever during the last month before the study.

Figure 3.Distribution of corrected MFI values against the P. vivax antigens in the L1, L2 and L3 groups.

Two risk factors were significantly and independently statistically associated with
a risk of having a high level of immune humeral response to P. vivax antigens:

– The distance to rivers (≤ 1.5 Km).

– The educational level (never schooled).

Geographical repartition

The different clusters of P. falciparum and P. vivax seroprevalence were presented in Figures
4,
5,
6, and
7 and in Additional file
9. For P. falciparum, the city of Djibouti showed a clustering of low and medium prevalence areas on both
sides of the Ambouli wadi. A hotspot was observed in Arhiba, in which more than half
of the population (56.4%) (Additional file
10) was seropositive for P. falciparum. A mean tendency was observed in the upper town (i.e., Quarters 1 to 15), which globally
showed a decreasing prevalence when the distance to Ambouli wadi increased. This tendency
was also observed in the lower town, i.e., the other side of the Ambouli wadi quarter,
with 4 hotspots (Balbala 2, PK12, Balbala 3, and North of Wahle Daba) that had similar
prevalence to Arhiba. The most prevalent cluster in the entire country was Balbala
4, in which almost two in three persons (67.5%) were seropositive for P. falciparum.

Figure 4.Map of clusters of Plasmodium falciparum seroprevalence in the capital of the republic of Djibouti.

Figure 5.Map of clusters of Plasmodium falciparum seroprevalence in the republic of Djibouti.

Figure 6.Map of clusters of Plasmodium vivax seroprevalence in the capital of the republic of Djibouti.

Figure 7.Map of clusters of Plasmodium vivax seroprevalence in the republic of Djibouti.

In the rest of the country (Additional file
11), the 2 regions in the north (Tadjourah and Obock) exhibited a low prevalence, except
for Balho, in which more than one in three persons were seropositive for P. falciparum. The situation in the South was more concerning, as 3 seropositivity hotspots were
observed in Dagguirou (46.9%), Tammiro (64.7%) and As-Eyla (64.7%) in the Region of
Dikhil, and 2 seropositivity hotspots were observed in Ali-Sabieh1 (52%) and Ali-Sabieh
3 (45.5%), the capital city of the Region of Ali-Sabieh.

Additional file 11.Map of clusters of P. falciparum and P. vivax seroprevalence in the Republic of Djibouti.

Considering the prevalence of P. vivax, in Djibouti city, on both sides of Ambouli wadi, an increase in the distance between
quarters and the wadi was associated with a decrease in prevalence rates, as was true
for P. falciparum. In the upper town, the only hotspot was in Arhiba, with a 33.3% seroprevalence.
In the lower town, two hotspots were observed in Balbala 4 and north of the Wahle
Daba, with 35% and 44.4% seroprevalence, respectively.

Of the northern regions, Obock and Tadjourah, the seroprevalence rates in some localities
were 20% to 30%, such as in Malâho, La’Assa (Obock Region) and the regional capital
Tadjourah (Tadjourah Region). In southern regions, the seroprevalence rates were between
20% and 30%, such as in Dagguirou, Garabayis (or Gour’abouss) and As-Eyla for the
Dikhil region and Doudoub Balaleh for the Ali-Sabieh region. Hotspots were observed
in Tammiro, with a 32.4% seroprevalence rate (in the region of Dikhil), and in Ali-Sabieh
1, with a 40% seroprevalence rate (the capital of Ali-Sabieh’s region).

Discussion

The present study was the first to analyse the P. vivax seroprevalence rate in the Republic of Djibouti. Supplemental information on the
P. falciparum situation in 2002 was also highlighted.

Serological tools for P. falciparum infection

The use of different antigenic peptides was dictated by the fact that in countries
where malaria transmission occurs, the results of serology may be ambiguous due to
cross reactions with other parasitic infections, such as toxoplasmosis
[26]. Thus, by increasing the number of antigens and considering the sera reaction to
at least two different plasmodial antigens, this enabled limiting the false positive
rates. However, this approach using solely antigenic peptides could lead to an underestimation
of the level of transmission; thus, other serologic markers were included, such as
the recombinant proteins MSP1 and AMA1, the use of which there is a consensus in the
literature
[27-29].

In 2009, Noor et al. observed a P. falciparum seroprevalence rate of 14.2% in adults above 50 years, 6.9% in children and an average
of 9.9% when they tested the reactivity of 4769 sera to one or both serological markers
PfMSP1 and PfAMA1 among Djiboutian population
[14]. The same method was applied to the present work and produced a seroprevalence rate
of 30.0%. Moreover, when the reaction to at least one marker of the 11 peptides, PfMSP1
and PfAMA1 were combined, the seroprevalence increased to 56.6%; finally, a seroprevalence
rate of 33.5% was obtained when considering reactions to at least 2 of the 13 markers.
In light of these observations, it can be deduced that the prevalence and, indirectly,
the P. falciparum malaria transmission have declined by at least a factor of three in the past seven
years. These results were consistent with the needs and obligations that lead to a
pre-elimination goal in which the reduction of transmission is the most important
key to pre-elimination
[15].

All obtained models predicting the P. falciparum or P. vivax seropositivity status or the level of humeral immune response to P. falciparum or P. vivax antigens have shown that female gender, a high educational level and never having
visited a malaria-endemic country more than one year were protective. Considering
the mixed infection seropositivity status, the multivariate logistic regression model
showed a protective effect of living at a distance > 1.5 Km from rivers and lakes,
in a rural area and not having had fever during the last month before the cross-sectional
study. Only the model that predicted the P. falciparum seropositivity status showed a protective effect of younger ages between 15 and 40
years. As serological tools reflect the cumulative exposition
[28], these observations suggest that the older populations were more exposed and that
transmission was thus higher in the past. In the city of Djibouti, educational level
is generally correlated with the level of wealth and therefore more accessibility
to health facilities and prevention measures
[30]. Historically, Ethiopia and Djibouti have maintained very important population exchanges
in both directions. Therefore, it is normal to see certain Djiboutian populations
settle there for long periods due to the far lower living costs when facing economic
or social difficulties in the Republic of Djibouti
[31]. Carteron in 1978 and Fox in 1991 have shown that Ethiopia was the most important
provider of malaria cases to Djibouti
[7,32]. This may explain the observation that living in malaria-endemic country (and especially
Ethiopia) for more than one year was a risk factor for being seropositive to P falciparum or P. vivax.

Geographical distributions of P. falciparum and P. vivax

In the city of Djibouti, the seroprevalence rates for both parasite species revealed
hotspots on both sides of the main wadi, i.e., Ambouli wadi and the quarters of Arhiba
and Balbala 4. Arhiba and Balbala 4 are quarters with significant migrant populations
who regularly travel to and/or from Ethiopia
[31]. There was an association between the decreasing of seroprevalent clusters to both
species and the increase of the cluster distance to Ambouli wadi.

In the rest of the country, P. falciparum seroprevalence rates were higher in the southern regions (Dikhil and Ali-Sabieh),
and in particular, hotspots were found along the land routes to Ethiopia, i.e., Tammiro/As-Eyla
and Ali-Sabieh. These roads are regularly used by professional truckers, private users
and migrants because they are the only two terrestrial roads to Ethiopia
[33].

Plasmodium vivax seropositivity status was more balanced throughout the Djiboutian territory, with
hotspots in the same locations in the southern regions as for P. falciparum. Because of the possibility of liver persistence of hypnozoïtes, P. vivax can spread more widely across the entire country in the case of incomplete treatment.
P. vivax can be found where P. falciparum is no longer detectable and can sometimes be more prevalent, as could be the case
in the neighbouring countries
[34]. This situation might explain the high seroprevalence rates recorded in northern
regions and suggests the occurrence of local transmission foci when Anopheles vectors exist.

The path to pre-elimination

Pre-elimination is a combination of technical feasibility, i.e., the baseline domestic
malaria transmission combined with the importation-related transmission, and operational
feasibility, which takes into consideration the country’s government status, health
status and information on populations at risk
[13].

Compared to one recent work
[14], the present serological study indirectly indicates that transmission may have decreased
by three-fold in the past seven years for P. falciparum, even though some hotspots were the same as those found in the Dikhil region. This
work, once combined with recent information provided by Noor et al. has shown that the Republic of Djibouti is likely on the correct path to pre-elimination
with benefits that are threatened by the persistence of hotspots such as those in
the Dikhil Region. Finding the same hotspots seven years later constitutes a serious
threat to the success of the announced goal. Taking into account technical and operational
feasibility, pre-elimination is possible only insofar as efficient control methods
are implemented at all administrative and executive levels of authority.

Recommendations emerge from the results of the statistical models. If the educational
level of the population cannot be increased, pre-elimination will require increased
awareness and health education for at-risk populations through methods that are appropriate
to local realities. Although being female seems to be protective, health education
should target mothers in particular to increase local knowledge, as has been previously
done with HIV
[35]. Regional collaborative alert systems are also indispensable because the population
is regularly moving to a neighbouring country. To act efficiently on hotspots, the
mass distribution of bed nets, mass chemotherapy and chemoprophylaxis for both parasites
and indoor residual spraying should be performed, followed by serosurveys and vector
monitoring. The vector monitoring system is actually debutant and in progress, although
it primarily concerns Djibouti city. Border posts should also see an improvement of
their monitoring activities and health controls.

Conclusions

As seen in this study, the P. falciparum seroprevalence rate was 25.90% in 2002, further studies with the same population
would be required to assess if there was a real decrease in transmission of P. falciparum in the Republic of Djibouti since 2002.

This is the first study that assessed the prevalence of P. vivax in the Republic of Djibouti. It is necessary to improve our knowledge of this pathogen
in order to create an effective elimination programme.

The protective effect of female gender, educational level and never having visited
a malaria-endemic area for more than one year was observed for both P. falciparum and P. vivax. For P. falciparum along, a protective effect was also observed for not having had a fever the last
month, living > 1.5 km away from lakes and rivers and being younger in age.

These findings should encourage authorities to improve efforts toward elimination
and to begin the final assault against the few persistent hotspots. However, to assess
the real pre-elimination status, the precise level of both P. falciparum and P. vivax transmission should be regularly monitored by serological methods or other tools
and including children less than five years old.

Consent

Blood samples were collected anonymously in accordance with the recommendations of
the Djiboutian Ministry of Health, which also gave ethical approval for the study.

Acknowledgements

We thank Dr. Chris Drakeley from the London School of Hygiene and Tropical Medicine,
London, UK, and Dr. Shirley Longacre from the Institut Pasteur à Paris, France, for
providing the PfAMA1, PfMSP1, PvMSP142 and PvMSP119 recombinant proteins. This study was supported by the Délégation Générale pour l’Armement
and the Direction Centrale du Service de Santé des Armées (grant no. 10co404 and grant
no.10co405).

We also thank Dr. Michel ETCHEPARE and Dr. Christian TOSI who supervised the collection
of blood samples with CR, and Dr. Mohamed Ali KAMIL of the Djiboutian Ministry of
Health, who facilitated the field studies funded by the World Bank.

Abramo C, Fontes CJ, Krettli AU: Cross-reactivity between antibodies in the sera of individuals with leishmaniasis,
toxoplasmosis, and Chagas' disease and antigens of the blood-stage forms of Plasmodium falciparum determined by indirect immunofluorescence.